Methods for Culturing Cells in an Alternating Ionic Magnetic Resonance (AIMR) Multiple-Chambered Culture Apparatus

ABSTRACT

Provided herein are methods for culturing cells, tissues or organoid bodies in the presence of a pulsating alternating ionic magnetic resonance field and models comprising a tissue-like assembly of the cells so cultured. The cells, tissues or organoid bodies are introduced into a culture unit comprising growth and nutrient modules in which the gravity vector of the growth unit is continually randomized and cultured in the presence of the alternating ionic magnetic resonance field.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional application claims benefit of priority under 35U.S.C. §119(e) of provisional application U.S. Ser. No. 61/686,690,filed Apr. 9, 2012, now abandoned, the entirety of which is herebyincorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of biophysics,bioelectromechanics, bioengineering, tissue engineering and cellularregeneration. Specifically, the present invention relates to analternating ionic magnetic resonance multiple-chambered cultureapparatus for potentiating or controlling the growth of biological cellsand tissues, such as mammalian tissue.

2. Description of the Related Art

Prior to the development of bioreactors, cell culture was limited tosystems subjected to the forces of gravity, with most laboratorycultures producing flat two-dimensional (2D), one cell thick specimensunlike the natural three-dimensional (3D) environment of a complex,multi-cellular organism. Most laboratory experiments therefore hadinherent limitations and a strictly one-dimensional view ofunderstanding how cells grew and interacted with one another in theirnatural environment.

With the development of bioreactors, most of these devices were “stirredtank” bioreactors that used a vertical aspect configuration with astirring device at the bottom of the growth chamber to mix the cells andfluid medium suspension. Horizontally rotating bioreactors offered a wayto minimize or neutralize the sedimentation and shear effects caused bygravity by using the “clinostat principal” in which a fluid medium wasrotated about a horizontal axis thus minimizing the wall effects andimpeller impacts of internal stirring devices and lowering the overallReynolds and Coriolis force effect on cells.

Cells grown in rotating bioreactors were suspended in a fluid medium andwere continually rotated away from the surfaces of the vessel whichenabled cells to adhere to one another and to grow. This type ofsuspended cell culture resembled growth mechanics in a naturallyoccurring tissue and in a multidimensional form and thereby promotedmore realistic, three-dimensional cell-to-cell contact signaling. These3D cells were induced to regulate and to produce cellular components asif grown within a complex organism and to produce complex matricescomprising extracellular matrix molecules, proteins, fibers, and othercellular components. These aforementioned processes lead toautoregulation and the ability to self-order in the human mammalianphysiology. Inside a complex organism, these components often informed acell of the neighboring environment and triggered a specific set ofresponses to that external environment. The cell grew or it becamestatic, which in turn, determined how the cell responded with theproduction of secondary regulators.

A typical rotating bioreactor had an outer tubular enclosure withtransverse end walls and end caps in the end walls. The outer tubularenclosure was supported on input and output shaft members androtationally driven by an independent drive mechanism. Coaxiallydisposed within the outer tubular enclosure was a central tubular filtermember that was rotationally supported on the input shaft and coupled tothe output shaft. The annular space between the inner and outer tubularmembers defined a cell culture chamber.

Two blade members were positioned about the horizontal axis and extendedlengthwise along the cell culture chamber. The blade members had radialarms at one end that were rotationally supported on the output shaft andradial arms at the other end that were coupled to the input shaft. Theinput shaft was rotationally driven by an independent drive means thatnormally drove the inner and outer tubular members and the blade membersat the same angular rate and direction so that no relative motionoccurred between these members. Thus, clinostat motion could be achievedfor the particles in the fluid within the cell culture chamber.

Existing bioreactors, however, are overly complicated systems and costlyto operate with respect to expenditure of preparation time, upkeep,disposal of non-reusable components. For example, in existingbioreactors the cell samples and any other required initial ingredientsmust be assembled into the culture system in preparation for anexperiment. At the end of the experiment, it is necessary to disassemblesignificant portions of the culture system in order to extract the cellsand/or tissue culture that grew during the experiment. Moreover, becausethe culture system mechanisms include rotating fluid couplings, leaksmay develop over time in the seals of these couplings. Finally, themotors that provide the rotation in existing bioreactors are integral tothe system, contributing to the complexity of the system and making itdifficult to maintain and operate the system. Accordingly, what isneeded is a culture system and method that mitigates or overcomes someor all of the shortcomings of existing bioreactors.

U.S. Pat. Nos. 6,485,963 and 6,673,597 disclose the use of atime-varying electromagnetic force (TVEMF) in a manner that stimulatesthe proliferation of cells grown in culture. In U.S. Pat. No. 7,179,217,Goodwin et al. disclose the use of a TVEMF sleeve for treatment of ananimal limb. Commercial utilization of this technology has provided twoapproaches to culture system design. The first approach is the use ofbaffles or plates within the culture system with a time-varyingelectromagnetic current applied across the plates to induce atime-varying electromagnetic force within the culture chamber. Thesecond approach is to use a coil wrapped around the rotating culturesystem chamber and affixed thereto with a time-varying electromagneticcurrent applied to the coil to create a time-varying electromagneticforce within the culture chamber.

There are several limitations with existing culture systems designs thatutilize TVEMF in the context of a rotating culture system chamber.First, the existing TVEMF culture systems have the electromagneticdevice permanently affixed to the culture chamber unit, which does notallow for the use of disposable modules nor does it accommodate theself-feeding capability of the current invention. Instead, existingsystems require periodic and frequent manual exchange of growth mediaduring the culture cycle. Additionally, since the goal of proliferationof cell cultures is in many instances the utilization of the cells andtissues for reintroduction into the human body for tissue regenerationor treatment of human maladies, the culture system chamber must meet therigid standards of the Food and Drug Administration (FDA). If the EMFinducing device is incorporated into the culture chamber, itsignificantly complicates the manufacture and sterilization process, andwould require routine disposal of the EMF inducing device along with theused culture system chamber. This would significantly add to the cost ofthe equipment and culturing process for FDA approved purposes.

Another limitation of existing culture systems is that they utilizeTVEMF, which does not effectuate the same stimulatory or physiologicaleffect on cultured cells as compared with alternating ionic magneticresonance. TVEMF fails to stimulate specific ionic species and membranechannel systems that play a major role in the regulation ofproliferation, differentiation, tissue repair, and related cellularmechanisms that are inherent to growth, development and maintenance of amammalian organism. A further limitation is that existing culturesystems rely on a batch fed or media perfusion systems to transfer mediainto and out of the growth chamber. Each of these methodologies fails toprovide physiological and homeostatic parameters similar to those of anaturally occurring physiological system.

Thus, there is a recognized need in the art for culture systems andmethods that utilize an alternating ionic magnetic resonance fieldduring the three-dimensional culture of cells, including tissues and/ororganoid bodies. Particularly, the prior art is deficient in methods forculturing cells, tissues and/or organoid bodies in an alternating ionicmagnetic resonance system comprising a culturing apparatus that utilizespre-sterilized and disposable modules and a removable alternating ionicmagnetic resonance chamber. The present invention fulfills thislong-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method for culturing cells. Themethod comprises introducing cells into a culture unit having a growthmodule and a nutrient module, randomizing continually the gravity vectorof the growth module and applying a pulsating alternating ionic magneticresonance field to the growth module during culturing of the cells. Thepresent invention is directed to a related method for culturing cellsfurther comprising the step of modulating the alternating ionic magneticresonance field to produce overlapping or fluctuating alternating ionicmagnetic resonance frequencies at one or more modal intervals spanningabout 6.5 Hz and ranging from about 7.8 Hz to about 59.9 Hz.

The present invention also is directed to a culture method for growingmammalian cells or tissues. The method comprises introducing mammaliancells or tissues into an alternating ionic magnetic resonance cultureapparatus. The alternating ionic magnetic resonance culture apparatuscomprises a nutrient module, having a proximal end and a sealable distalend, that contains a nutrient media, a growth module, having a proximalend and a distal end, that contains the cells or tissue and that isfilled with nutrient media and sealed to remove observable gases, saiddistal end of the growth module fluidly sealed to the proximal end ofthe nutrient module, a randomizing adapter electrically connected to arandomizing mechanism and containing the nutrient module therein, and anelectromagnetic chamber comprising a conductive wire to produce apulsating alternating ionic magnetic resonance field. The gravity vectorof the growth module is randomized continually with the randomzingadapter and the pulsating alternating ionic magnetic resonance field isgenerated around the growth module during culturing. The presentinvention is directed to a related method for culturing cells furthercomprising the step of modulating the alternating ionic magneticresonance field to produce overlapping or fluctuating alternating ionicmagnetic resonance frequencies at one or more modal intervals spanningabout 6.5 Hz and ranging from about 7.8 Hz to about 59.9 Hz.

The present invention is directed further to a model comprising atissue-like assembly of the cultured cells or tissue produced by themethods described herein.

Other and further aspects, features and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate preferred embodiments of the invention and therefore are notto be considered limiting in their scope.

FIGS. 1A-1C is an overview of the unassembled (FIG. 1A), partiallyassembled (FIG. 1B) and assembled (FIG. 1C) primary components of theculture apparatus comprising a culture unit, a randomizing adaptor and aremovable adjustable alternating ionic magnetic resonance module.

FIGS. 2A-2B are front (FIG. 2A) and back (FIG. 2B) views of the growthmodule.

FIG. 3 illustrates the assembly of the growth module with the nutrientmodule to form the culture unit.

FIG. 4 illustrate culture cell viability parameters of HBTC cells grownwith alternating ionic magnetic resonance. Growth module samples weretaken prior to weekly changing of the media in the nutrient module andmeasurements of glucose utilization and culture pH v. time were made.

FIG. 5 is a cell growth and tissue assembly curve for HBTC cells withand without exposure to alternating ionic magnetic resonance over atwenty day growth period.

FIGS. 6A-6D are calcium and potassium ion transport micrographs of HBTCcells grown in alternating ionic magnetic resonance culture apparatus.FIG. 6A depicts calcium ion staining of HBTC cells grown alone exposed(left) and unexposed (right) to alternating ionic magnetic resonance.FIG. 6B depicts calcium ion staining of HBTC cells grown on cultispheremicrocarriers exposed (left), unexposed (right) to alternating ionicmagnetic resonance and microcarrier control treated with Fura 2AM. FIG.6C depicts potassium ion staining of HBTC cells grown on cultispheremicrocarriers exposed (left) and unexposed (right) to alternating ionicmagnetic resonance. FIG. 6D depicts potassium ion staining of HBTC cellsexposed to alternating ionic magnetic resonance (top), grown without anelectric field (middle) and control microcarriers alone (bottom).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “a” or “an”, when used in conjunction with theterm “comprising” in the claims and/or the specification, may refer to“one”, but it is also consistent with the meaning of “one or more”, “atleast one”, and “one or more than one”. Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the invention. It is contemplated thatany device or method described herein can be implemented with respect toany other device or method described herein.

As used herein, the term “or” in the claims refers to “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or”.

As used herein, the term “about” refers to a numeric value, including,for example, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term “about” generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited value) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). In some instances, the term“about” may include numerical values that are rounded to the nearestsignificant figure.

As used herein, the terms “proximal” and “distal” refer to components orparts thereof or fields that are nearer or farther from the growthmodule, respectively. With respect to the growth module per se, proximalrefers to the side comprising the gas membrane and distal refers to theside comprising the baffling that engages with the nutrient module.

As used herein, the term “animal” refers to a mammal, preferably ahuman.

In one embodiment of the present invention there is provided a methodfor culturing cells, comprising the steps of introducing cells into aculture unit having a growth module and a nutrient module; randomizingcontinually the gravity vector of the growth module; and applying apulsating alternating ionic magnetic resonance field to the growthmodule during culturing of the cells. Further to this embodiment themethod comprises modulating the alternating ionic magnetic resonancefield to produce overlapping or fluctuating alternating ionic magneticresonance frequencies at one or more modal intervals spanning about 6.5Hz and ranging from about 7.8 Hz to about 59.9 Hz. Particularly theoverlapping or fluctuating alternating ionic magnetic resonancefrequencies produced are about 10, 14, 15, 16, or 32 Hz.

In one aspect of both embodiments the introducing step may compriseplacing the cells into the growth module; filling the growth module withgrowth media; sealing the growth module to remove observable gases;attaching the growth module to the nutrient module; and adding freshgrowth medium to the nutrient module. In another aspect of bothembodiments the randomizing step may comprise positioning the nutrientmodule comprising the culture unit into a randomizing adapter; andrandomizing the gravity vector via a randomizing mechanism comprisingthe randomizing adapter. In yet another aspect the applying step maycomprise positioning the growth module comprising the culture unit intoan electromagnetic chamber and generating the pulsating alternatingionic magnetic resonance field in the electromagnetic chamber.

In all embodiments and aspects thereof the nutrient module may comprisean open-ended body having a proximal end with a diameter of a length toreceive the growth module therein; a first gas-permeable membrane with agas port disposed thereon comprising the proximal end; and a distal endcomprising a first sealable opening. Also the growth module may comprisea body having a proximal end comprising a second gas-permeable membranewith a plurality of inlet/outlet ports disposed thereon; and a distalend comprising a baffling system and a semi-permeable membrane in fluidcontact with the first gas-permeable membrane. Particularly, the growthmodule and the nutrient module are disposable. In addition the pulsatingalternating ionic magnetic resonance field may produce one or more of amodification, growth, differentiation, dedifferentiation, altered geneexpression, altered transcription events or cellular regeneration viacontrolling the ionic transport or transcription mechanism in the cells.Furthermore the alternating ionic magnetic resonance field may changethe mammalian cellular ionic transport thereby enabling infection and anexpression of mammalian viral epitopes. Further still the cells maycomprise a tissue, an organoid body, a virally-infected cell, or abacterially-infected cell.

In another embodiment of the present invention there is provided aculture method for growing mammalian cells or tissues, comprising thesteps of introducing mammalian cells or tissues into an alternatingionic magnetic resonance culture apparatus comprising a nutrient module,having a proximal end and a sealable distal end, that contains anutrient media; a growth module, having a proximal end and a distal end,that contains the cells or tissue and that is filled with nutrient mediaand sealed to remove observable gases, the distal end of the growthmodule fluidly sealed to the proximal end of the nutrient module; arandomizing adapter electrically connected to a randomizing mechanismand containing the nutrient module therein; and an electromagneticchamber comprising a conductive wire to produce a pulsating alternatingionic magnetic resonance field; randomizing, continually, the gravityvector of the growth module with the randomzing adapter; and generatingthe pulsating alternating ionic magnetic resonance field around thegrowth module during culturing. Further to this embodiment the methodcomprises modulating the alternating ionic magnetic resonance field, asdescribed supra.

In both embodiments the nutrient module may comprise a firstgas-permeable membrane with a gas port disposed thereon comprising theproximal end. Also the growth module may comprise a second gas-permeablemembrane with a plurality of inlet/outlet ports disposed thereoncomprising the proximal end and a baffling system and a semi-permeablemembrane comprising the distal end in fluid contact with the firstgas-permeable membrane. Particularly, the growth and/or the nutrientmodules may be disposable and the materials comprising the alternatingionic magnetic resonance culture apparatus may be sterilizable. Inaddition wherein culturing in the pulsating alternating ionic magneticresonance field produces one or more of a modification, growth,differentiation, dedifferentiation, altered gene expression, alteredtranscription events or cellular regeneration via controlling the ionictransport or transcription mechanism in the cells or tissues.Furthermore, culturing in the alternating ionic magnetic resonance fieldmay change the mammalian cellular ionic transport thereby enablinginfection and an expression of mammalian viral epitopes. Particularly,the mammalian cells or tissues are virally or bacterially infected.

In yet another embodiment of the present invention there is provided amodel comprising a tissue-like assembly of the cultured mammalian cellsor tissue produced by the methods as described supra.

The present invention provides methods for the short and long-termproliferation, growth, enrichment, conditioning, modification, and/oraggregation of mammalian cells, tissues or organoid structures inadaptable culture systems in alternating ionic magnetic resonancefields. alternating ionic magnetic resonance fields comprise specificbio-electromagnetically relevant frequencies that mimic the globaldiurnal cycle believed to influence cellular behavior and geneticevolution. Particularly, an alternating ionic magnetic resonance field,such as produced in the alternating ionic magnetic resonance chamberpresented herein, mimics in part the natural environment that mammaliancells are exposed to in the normal earth-based living system.

Thus, the present invention provides methods for three-dimensionalgrowth of a culture material, such as, but not limited to, animal,preferably mammalian, cells, tissues, organoid bodies, etc. The culturematerial is introduced into the growth module and grown in thenutrient-rich media provided by the nutrient module in the presence ofan alternating ionic magnetic resonance field. During growth the gravityvector of the culture unit is randomized to favor three-dimensionalgrowth. This maximizes the efficiency of metabolic exchange within thesystem while simultaneously providing for the accumulation of valuablebiomolecules in the growth module and the nutrient module. A morecontrolled cell growth culture system is thus enabled that can bemanipulated to provide for increased rate of cell growth, fasterdifferentiation, increased cell fidelity, and the induction orsuppression of selective physiological genes involved in directingcellular differentiation.

Specific culture material may be selected and conditions set toregulate, for example, gene expression and protein activity within thecultured material. The alternating ionic magnetic resonance fieldstimulates the expression and regulation of various genes, includingtranscription factors, and alters the activity of the genome. Thisresults in a modified output of existing cellular proteins, such as celltransport proteins involved in regulating ionic concentration, membranetransport and other crucial pathways in the regulation of growth,development, and differentiation, dedifferentiation, cell maintenanceand aging-related mechanisms in animals and plants. Regulating celldifferentiation/dedifferentiation via the methods and processes providedherein may facilitate the development of faster healing and lifespanextension compositions and applications.

In systems where it is desired to have the cells adhere to substratesfor the sole purpose of proliferation without promotingthree-dimensional tissue growth, the cells can be grown directly on aflat, two-dimensional electrode surface composed of a biocompatiblematerial. In these situations, some cultured cells may actually beattracted to the supportive electrode material, coatings, orelectrically conductive channels that can be incorporated into theculture unit to facilitate cell attachment. For example, the alternatingionic magnetic resonance field is induced in the region of the channelby passing the alternating ionic magnetic resonance protocol through aconductor placed along the channel.

In some systems microcarrier spheres or beads are included and suspendedwithin the culture medium to induce adherence of the cells to the beads.For cell proliferation in these systems, the culture system growthmodule is preferably exposed to the randomization at a range of about 2to 60 rpm, and the alternating ionic magnetic resonance is generated bya time-varying current passed through a conductor with an RMS value ofabout 0.001 to 10,000 Gauss with a preferred range of about 0.01 to 3000Gauss.

Particularly, the present invention provides methods for up-regulatingor increasing viral replication and proliferation genes and geneproducts in a culture material, such as, cells, tissue, etc. Culturematerial infected with a virus of interest, grown in the alternatingionic magnetic resonance culture system induces the up-regulation ofgenes associated with the virus, particularly, those associated withreplication and proliferation. In non-limiting examples, such models andsystems would be useful for producing large numbers of virions forvaccine production, identification of viral genomic adaptation products,tracking of viral genomic shift during a long term culture, developmentof antivirals or antibacterials targeted at blocking replication, andharvest of human cell-produced proteins that only result from virally orbacterially infected cells. Correspondingly, the methods provided hereinare applicable to culture materials infected with or grown with abacteria of interest to up-regulate bacterial-associated genes. Methodsof identifying proteins or other products from the media comprising theculture system are well-known in the art as are methods for developmentof antivirals and an antibacterials based on specific compounds,proteins, nucleic acids, etc.

Moreover, the present invention provides models of 3-dimensionaltissue-like assemblies (TLAs) of cells. The tissue-like assemblies arestable for at least 3 months, preferably 6 months or longer and sharefeatures with the corresponding 2-dimensional tissues/cells or withtissue/cells obtained in vivo. The cells may be grown with or without analternating ionic magnetic resonance field. Alternatively, the model maycomprise tissue-like assemblies of cells infected with a virus or abacteria, particularly a pathogen. These tissue-like assemblies alsoremain stable for at least three months. Moreover the viral or bacterialgenome remains stable throughout the infection period. Such models areuseful for, but not limited to, the up-regulation of viral or bacterialassociated or induced genes and/or gene products and/or the study ofviral or bacterial adaptive mechanisms.

Generally, the culture system comprises a gravity randomizing,multiphasic culture system having a disposable self-feeding growthmodule, a nutrient and growth module that comprises a culture unitwhich, optionally, is disposable, and a removable electromagneticchamber or unit which, when applied to the outside of a culture unit, issuitable for delivering alternating ionic magnetic resonance fields tothe contents of the culture unit. Existing culture systems using PEMFand TVEMF have been shown to increase the rate of cell growth of thecells cultured in the system. The alternating ionic magnetic resonanceculture system is a significant improvement on these systems andincorporates instead the use of an alternating ionic magnetic resonancedevice which induces cell regeneration, increases cell fidelity,modulates cellular transcription and induces the selective regulation ofkey physiological genes useful in directing the differentiation anddedifferentiation process of particular cells.

The generated alternating ionic magnetic resonance field, as describedherein, produces a series of controlled resonating waveforms that mimicthe Schumann Resonances, which are global electromagnetic frequenciesthat are excited by lightning discharges, with more precision than arecreated naturally. The alternating ionic magnetic resonance-generatedresonating waveforms can be modified or accelerated to specificallyregulate or induce a physiological response in a particular cell system,i.e., a pulsed emission to preferentially effect the oscillation ofspecific ion species in the living cell. Each cell type has thepotential to respond to a given resonating pattern differently thananother cell type based on total ion content and ion species. Moreover,each physiological response may involve the induction of differentcellular control mechanisms, such as, but not limited to, stimulated ordecreased genomic, proteomic, transcriptomics, and metabolomicexpressions, altered ion flow through the membrane, and altered genereplication.

Thus, the alternating ionic magnetic resonance culture system andapparatus and methods for use can stimulate the expression andregulation of various genes, including transcription factors, and toalter the activity of the genome to result in modified output ofexisting cellular proteins, such as cell transport proteins involved inregulating ionic concentration, membrane transport and other crucialpathways in the regulation of growth, development, and differentiation,dedifferentiation, cell maintenance, inflammation, and aging-relatedmechanisms in animals and plants. Use of the alternating ionic magneticresonance culture system, apparatus and the methods to stimulate geneexpression and regulation, is relevant to both practical commercialapplications as well as applications relating to investigations thatfocus upon re-creating initial conditions in the context of evolutionarybiological processes at the cellular and physiological level.

The alternating ionic magnetic resonance culture system and apparatusalso offers the ease and convenience of using disposable components forready compliance with rigid FDA requirements addressing cleanliness andthe avoidance of cross-contamination of cell species. The use of adisposable culture unit facilitates the manufacture and use of a systemthat can easily meet the strict requirements of the FDA. Components canbe manufactured and packaged in sterile packs for ready use by one ofordinary skill in the art, much the same as other disposable medicaldevices are used. The alternating ionic magnetic resonance chamber ofthe current invention facilitates selective reuse of the ionic magneticresonance (IMR) device, which contributes to minimizing the costsassociated with culturing cells and tissues for medical purposes.

The alternating ionic magnetic resonance culture system comprises aculture unit, which has a pre-sterilized, disposable, self-feedinggrowth module and a pre-sterilized disposable nutrient module, aremovable and interchangeable alternating ionic magnetic resonanceelectromagnetic chamber, and a means for continually randomizing thegravity vector of the growth module and nutrient module, such as arandomizing adapter. The pre-sterilized and disposable componentsminimize cumbersome handling, costs and difficulties associated with theimproper delivery of the IMR fields in known culture systems and EMF andTVEMF designs. Alternatively, the growth and nutrient modules maycomprise reusable materials and the alternating ionic magnetic resonancechamber may be disposable.

The randomizing adapter holds the culture system in a horizontalposition, whereby a basically cylindrical culture system can rotate ormove clockwise and/or counter clockwise horizontally about its centralradial axis to minimize adherence of the cells to the reactor walls. Therandomizing adapter comprises a randomizing device or mechanism forcontinually randomizing the gravity vector in the growth module orculture system alone within a stationary nutrient module and astationary electromagnetic device, in unison with an electromagneticdevice located inside a stationary nutrient module or together with thenutrient module and electromagnetic device.

A continuously randomized culture system provides a three-dimensionalgrowth environment effectuated by continual gravity randomization,steady but consistent disruption, such as oscillation. Minimalturbulence randomization discourages adherence of eukaryotic cells tothe walls of the culture system while encouraging self-adherence of thecells to one another. The randomizing adapter accommodates rotations oroscillations of at least the growth module sufficient to minimizeadherence of the cells to the walls of the chamber. Different cell typeshave different adherence factors, so depending on the type of cells tobe cultured, the optimal rate of rotation will fluctuate. The adherencefactor will also become more important as the cells proliferate withinthe chamber and become more concentrated, whereby there is moreinteraction with the wall of the chamber. As such, the rate of rotationor oscillation of the chamber often increases as the density of cellsincreases in longer runs in the culture system. Consequently, thecontinually randomized gravity vector device optimally comprises avariable setting that can accommodate growth chamber rotation speeds inthe range of 0.01 to 60 rpm, with a preferred range of 2 to 40 rpm. Insystems using an oscillating type of device, periodic oscillations mayrange in frequency from being continuous to oscillating every 30 secondsor even every half hour.

The randomizing adapter may be a simple system of external rollers onwhich the culture system sits, similar to typical tissue culture rollerbottle mechanisms. Alternatively, rotation can be effectuated by anexternal electric motor using a system of fan-belt like connectionmechanisms or a direct drive. The randomizing mechanism maysystematically rotate the entire culture system or the culture unit orthe growth module alone. The type of rotation device will dictate thetype of adapter necessary on the component parts, such as a pulley-likewheel that would be firmly attached to a spindle incorporated into tothe affixed growth module cap and extends through a sterile liquid tightadapter in the nutrient module cap.

The growth module contains a small volume of culture media, as well asthe cells and/or tissue and, optionally, a matrix material to becultured, that completely fills the module with no noticeable air space.It has an integral semi-permeable molecular membrane incorporated intoone of its walls to facilitate the diffusion of gases, nutrients andwastes between the cell culture chamber and the extra nutrient-richmedia in the surrounding nutrient module. The molecular membrane of thegrowth module contains a diffusible osmotic membrane capable ofexclusion thresholds from 100-500,000 MW with a preferable cutoff rangeof 2000-12500 MW. The osmotic semi-permeable membrane is generallycomposed of a hydrophilic composition, but may comprise a morestructural composite coated with a hydrophilic composition (e.g.nitrocellulose, polysulphone, polyacetate, or other similar composite).Unlike a perfused system, the semi-permeable membrane system facilitatesthe transport of nutrients and wastes without the loss of valuablebiomolecules from the growth module. The retention of these biomoleculesincreases the accuracy and fidelity of the mammalian organoidrecapitulation. Additionally, the specific membrane exclusion cut offprovides a means to enhance the production of valuable cellularproteomics. This enhancement saves time, effort and purification costs.The growth module also comprises means for securement

The nutrient module may be disposable and serves as a media reservoirthat attaches to or surrounds the growth module. The nutrient module hasat least one sealable opening at one end, which is sealable with anappropriate cap, e.g., screw-top, snap-top, crown-top, crimped-top,slide-top, and designed to be large enough to insert an appropriatelysized growth module therein. The cap may have an adapter assembly forconnecting an external movement device capable of delivering a continualrandomized movement to the growth module or culture system, for example,oscillating or rotating in a mono- or bidirectional manner. Preferably,the entire culture system is attached via the wall of the nutrientmodule to a bidirectional motor device that slowly randomizes thegravity vector of the entire system.

The nutrient module supplies a continually diffusible supply of freshmaterial to the cultured cells and is adapted with a gas port or gasexchange vent fitted with a semi- or gas-permeable membrane to providefor the exchange of waste gases. Carbon dioxide and ammonia generated bythe tissues in the growth module diffuse out of nutrient module andatmospheric, i.e., 159 mm Hg, oxygen diffuses into the nutrient modulethrough the gas-permeable membrane of the gas port. The gas permeablemembrane may be a dialysis membrane, a thin gas-permeable siliconemembrane or a similar material. The gas port may be incorporated intothe nutrient module cap for convenience or may be located in the wall ofthe nutrient module as a separate opening to the outside environment.The nutrient module includes a mixing device located externally to thegas permeable membrane.

These processes maintain homeostatic physiological conditions in theculture much as see in the human or mammalian body. The nutrient moduleis large enough to accommodate the full volume of the growth module inaddition to a sufficient volume of media to effectuate efficientexchange of nutrients and oxygen from the fresh media to the growthmodule and waste products and gases away from the growth module forremoval from the system.

The growth module may be disposable and/or an internal module and istypically a cylindrical container, although it may be any shape, suchas, but not limited to, a sphere or bag. The growth module has asealable opening at one end that is fitted with and sealed with anappropriate sterile, liquid-tight cap, for example, screw-top, snap-top,crown-top, crimped-top, slide-top, that may have one or more ports foreasy assembly, injection, inoculation and harvest. The sterileliquid-tight cap provides also for the growth module cap to fit into aliquid-tight randomizing adapter that allows for rotation of the growthmodule. The growth module may be adapted with inlet and outlet ports forthe periodic or continual exchange of media through the chamber and maybe equipped with a baffling system that efficiently directs a slowcontinual flow of fresh media and nutrients across the osmotic membraneto allow more control over nutrient transport between the modules to aidin maintaining a more controlled, homeostatic environment. Such abaffling system streamlines the use of fresh media and has the potentialof decreasing the overall amount of media needed during the course of aculture experiment.

One wall of the growth module at least partially comprises asemi-permeable, hydrophilic dialysis membrane that contains the cellsand/or tissue within the confines of the growth module while allowingthe free diffusion of gases, nutrients and metabolic wastes with thefresh media in the nutrient media-hold compartment. A second wall in thegrowth module comprises a gas permeable membrane that is hydrophobic andcontrols the resident dissolved gas coefficient in the growth module.

The dialysis membrane may be any material with pores large enough forthe transfer of small molecules, but small enough to retain intact cellswithin the growth module. It may comprise a gas-permeable siliconecomposition or a polyethylene type material that provides for efficienttransport of carbon dioxide, dissolved in the culture medium, both as agas and as a solute in the form of sodium bicarbonate from the growthmodule to the nutrient module and for the transport of oxygen into thebioreactor. The dialysis membrane may be covered with an additionalsupport or membrane stabilizer that protects the dialysis membrane frommechanical damage during handling, setup and harvest, as well as duringthe culture stage to prevent damage from moving or swirling media in themodules.

In addition to permitting transport of carbon dioxide and oxygen, thedialysis membrane is selected to have a pore size sufficient to permitthe diffusion of other solubilized nutrients, such as sugars, aminoacids, vitamins, ions, etc., from the fresh media in the nutrient moduleto the growth module, as well as the transfer of metabolic byproducts,such as acidic compounds, for example, lactic acid, toxic gases, e.g.carbon dioxide, toxic solutes, e.g. ammonium ions, and other lowmolecular mass products from the growth module to the nutrient module.The pore size must be small enough, however, to prevent the transfer ofcells and high molecular weight cell products, such as, secretedproteins, antibodies, glycoproteins, large nucleic acids, etc., into thenutrient module.

The growth module may be disposed inside a larger nutrient module. Thegrowth module may be filled with cells/tissue and media, and sealedprior to insertion into the nutrient module, or may be inserted emptyand assembled inside the nutrient module, and later filled and sealedwhile inside the nutrient module. The ability to remove the growthmodule and move it to another nutrient module facilitates subsequentprocessing of the cultured cells/tissues with minimum hazard ofcontamination and loss of time and efficiency.

In one non-limiting example of a culture unit, a 35 ml or 50 ml capacitygrowth module is fitted to a 450 ml nutrient module by a snapping orother connection mechanism or means. The outer nutrient module holdssufficient media to provide support of cell growth inside the smallergrowth module for a period of several days or more. In anothernon-limiting example of a culture unit, the nutrient module issignificantly and substantially larger than the growth module wherebythe volume of the media-hold compartment now exceeds the volume of thegrowth module by as much as 100,000 fold. For instance, one or multiplesmall 1-5 cc growth module(s) may be completely submersed in a 100-liternutrient module (similar to a 25-30 gallon bacterial fermentation tank),or one or more rod(s) comprising multiple tandem units of smaller 1-5 ccgrowth modules may be submersed in an elongated cylindrical nutrientmodule. This is more conducive for periodic manual exchanges of media.

Preferably, a larger nutrient module having a volume 2 to 50 times thatof the growth module is used. With larger nutrient modules, manualexchange of the media at periodic time intervals without having tocontinually feed fresh media into the module is possible. As such,culture units having larger nutrient modules need not have inlet andoutlet ports for media exchange, but, optionally, may have one or moresets of ports for convenience in handling the media. Culture unitsrequiring periodic manual exchange of the media would preferably havenutrient module volumes greater than 10 times that of the growth module.

The growth and nutrient modules may be made of disposable biocompatiblepolycarbonate based materials that can be autoclaved under controlledconditions for reuse if necessary, or they may be made of more durablecomponents such as glass or stainless steel or polycarbonates/plastics.The growth module comprising the dialysis membrane is more adapted toirradiation type sterilization and better for prepackaged blister-likemanufacture and sterilizing. The nutrient module can be reused and maybe made of polycarbonate or a more stable material such as glass orstainless steel.

The alternating ionic magnetic resonance chamber comprises anelectromagnetic modulating device configured to deliver a pulsatingalternating ionic magnetic resonance field to cultured cells/tissue,organoid bodies, etc. within the growth module. The electromagneticdevice may comprise an electrode or set of electrodes or a removablechamber that is easily interchangeable depending upon the needs of thesystem. Preferably the alternating ionic magnetic resonance chamber isan easily removable chamber that encompasses or fits around or receivestherein the entire culture system or only the growth chamber. In theform of a removable chamber, the chamber is a slip-on chamber that holdsa coil designed to be larger than the diameter of the culture unit. Itis made of a relatively rigid electrical conductive material, e.g., awire, wound in a cylindrical or rectangular shape that when connected toa pulsating electromagnetic current creates a electromagnetic force inthe range of about 0.001 to about 10,000 Gauss within the internalportion of the chamber and the encompassed culture device.

Preferably, the conductive wire of the alternating ionic magneticresonance chamber is made of a conductive ferromagnetic material coiledabout an electromagnetic permeable polymer at about ten coils per inch.The coil can be encased in a thin flexible encasement made of a smoothconductive material that provides for easy handling during assembly anddisassembly of the culture system and convenient cleaning before andafter use. Also, the alternating ionic magnetic resonance field may begenerated by a device producing a pulsating time-varying current passedthrough a conductor with an RMS value of about 0.01 to about 10000 mA,with a preferred range of about 1 to about 5000 mA for some cellsystems.

The alternating ionic magnetic resonance protocol/signal can begenerated by many commercially available devices that are commonlyreferred to as random/arbitrary waveform or waveform generators, such asunits produced by Tektronics, e.g., models AFG3021B, AFG3022B, AFG3101,AFG3102, AFG3251, AFG3252; and Agilent, e.g., models 33220A, 33250A, and33220A-HO1, among numerous other suppliers. The waveform generator isprogrammed to produce the desired series of pulses at the desiredfrequencies over a specific time interval. This signal is then connectedto the output or transmission device either directly or though anamplifier to strengthen/regulate or increase the intensity of the fieldif desired. Alternatively, the “signal or waveform protocol” isprogrammed onto a custom designed computer chip and the series ofdesired signals are emitted from the chip to the transmission deviceafter it is energized via a power supply that will produce the desiredfield strength in the transmission device.

The alternating ionic magnetic resonance field is a multivariant fieldand may be induced by either a multi varying current within a conductoror by a multi varying voltage between fixed conductors. For example, theculture is placed near a conductor through which a time-varying currentis passed. Alternatively, the culture is placed between parallel platesupon which a time-varying voltage is applied. In both cases, analternating ionic magnetic resonance results within the region of thecell culture.

Several methods can be used to produce an alternating ionic magneticresonance signal, such as delta or square wave, Fourier curve or acombination of signals within a given time domain. For example, an arrayof conductive current carrying (voltaic) electrodes can be arranged tofocus the electromagnetic (EM) field in the specific chamber holding aculture. An alternating ionic magnetic resonance can also be applied toenhance tissue growth that may occur on a shaped or custom designedsubstrate within the chamber. The electromagnetic field may be generatedby various means, such as, by directing the current waveform directlythrough a conductive substrate or substrate layer or by projecting thefield from an external electrode, for example, a plate, an antenna, acoil, or a chamber, or from a set of electrodes adjacent to and spacedapart from, but in the immediate vicinity of, the medium, so that therelative strength of the electromagnetic field is effective within thegrowth chamber. For example, a current of about 100 milliamps, conductedbetween opposite corners of a metallic conductor, produces a stimulatoryalternating ionic magnetic resonance extending several centimeters fromthe plate surface.

Particularly, when the alternating ionic magnetic resonance field isgenerated through conductive antennae, external or in direct contactwith the media, e.g., wire, electrode, coil or similar transmissiondevice, the field is adjacently spaced apart from the cultured cells andmedia and carries an alternating ionic magnetic resonance signaladvantageously produced by a varying electrical potential in the form ofa delta or square wave having the preferred fundamental frequencies ofapproximately 10-300 cycles per second (Hz). Particularly, one or moreoverlapping or fluctuating alternating ionic magnetic resonancefrequencies at fundamental intervals of 10, 14, 15, 16, or 32 Hz, and,optionally, resonances that fluctuate between about 8 and 14 Hz (roundedvalues) can be produced and passed through the antennae or transmissiondevice. The fundamental intervals include the respective harmonicintervals extending to 256 Hz, and incorporating all harmonics of theaforementioned fundamental frequencies to infinity in the form of asquare wave of 0.01-10000 mA with a nearly zero time average

Preferably, a two-dimensional or a three-dimensional directionalantennae may be utilized and may be applied to conventionaltwo-dimensional or to three-dimensional tissue cultures.Three-dimensional cultures may be achieved in actual microgravity or bycontinually randomized gravity vector vessel technology that simulatessome of the physical conditions of microgravity, and/or in other,conventional three-dimensional matrix based cultures. Theelectromagnetic field, preferably an alternating ionic magneticresonance field, is achieved in the vicinity of the antennae or coil bypassing, through the directional device, a pulsating electromagneticfield of the correct frequency, duration, and field strength, for theproper duration.

During use of the culture system the range of frequency and oscillatingelectromagnetic field strength is a parameter that may be selected toachieve the desired stimulation of the cultured material, such astissues, cells or genes, etc. of interest. The final field produced canbe in the range of 0.001 to 10000 Gauss, but the preferred range insidethe central region of the chamber cylinder and the growth module of theculture system is in the range of about 0.01 to about 5000 Gauss.

A preferred embodiment of the alternating ionic magnetic resonanceculture system is depicted in the figures and described below. However,such reference is not meant to limit the present invention in anyfashion. The embodiments and variations described in detail herein areto be interpreted by the appended claims and equivalents thereof.

As shown in an unassembled view in FIG. 1A, the alternating ionicmagnetic resonance culture apparatus 100 comprises a randomizing adapter110, a culture unit 120, and an AIMR chamber 150. The randomizingadapter has an open, circular proximal end 112 with a diametersufficient to accommodate the culture unit therein and a distal end 114in electrical communication with a randomizing mechanism 116. Theculture unit comprises a growth module 130 at the proximal end and anutrient module 140 at the distal end of the culture unit into which thegrowth module is fitted and secured at least via a securing or fasteningmeans 138 a,b to a lip, rim or edge 141 a comprising the proximal end141 of the nutrient module. The growth and nutrient modules are shownhere comprising substantially cylindrical bodies, however the modulesmay have other shapes as long as the nutrient module can securely andfunctionally accommodate the growth module and contain nutrient mediatherein and the growth module can securely and functionally contain aculture material for growth therein and receive and exchange nutrientmedia and gases.

The growth module 130 has a front or proximal wall 131 comprising a gasmembrane 132 and inlet and outlet ports 133 a,b,c disposed through thegas membrane (see FIG. 2A). The back or distal wall of the growth modulecomprises a baffling means or system 136 which when affixed to theproximal end 141 of nutrient module is in fluid communication therewith(see FIG. 2B). The proximal end 141 of the nutrient module comprises agas port or gas exchange vent 145 fitted with a semi- or gas-permeablemembrane 146 (see FIG. 3). The distal end 142 of the nutrient modulecomprises a cap 143 covering an opening 144 into the nutrient modulethat is adaptable to engage with the randomizing mechanism. Optionally,the nutrient module may comprise a means for indicating media volume 147etched or disposed on the module surface. The AIMR chamber 150 hascircular proximal 151 and distal 153 ends with a diameter sufficient toslide or fit over the culture unit and comprises an electromagneticdevice 155, in this instance a coil, disposed around the exteriorthereof and means or device 157 for generating a pulsating, time-varyingelectromagnetic current (PTVEC) in electrical communication with theelectromagnetic device.

FIG. 1B illustrates how the culture unit is accommodated within therandomizing adapter. The distal end 142 of the nutrient module 140comprising the culture unit 120 is disposed within the proximal end 112of the randomizing unit 110 and is electrically engaged with therandomizing mechanism 116 (not shown). This leaves the growth unit 130uncovered and available to receive an alternating ionic magneticresonance field. Assembled, as shown in FIG. 1C, the proximal end 151 ofthe AIMR chamber 150 is disposed around the proximal end 141 of thenutrient module, particularly such that at least the growth module 130is disposed within the alternating ionic magnetic resonance chamber toreceive the alternating ionic magnetic resonance field generated by theelectromagnetic device 155. A pulse sensor 159 is disposed on theelectromagnetic device.

With continued reference to FIGS. 1A-1C, FIG. 2A is a front view of thegrowth module 130. The front or proximal side 131 of the growth modulecomprises a gas membrane 132 disposed across the surface thereof. Thegas membrane comprises a plurality of protusions, generally representedby 134 a,b,c,d,e,f, radially disposed across the surface of the membraneto increase the surface area and has a plurality of inlet/outlet portsrepresented as 133 a,b,c disposed through the membrane and in fluidcommunication with nutrient media contained within the growth module.

FIG. 2B is a back view of the growth module 130. The back or distal side135 comprises a baffling system 136 disposed therein and asemi-permeable dialysis membrane 137 comprising at least part of thedistal side. The gas-permeable membranes 132 and 146, including theinlet/outlet ports 133 a,b,c and the gas port 145 are in fluid contactwith the nutrient media in both the growth module and the nutrientmodule. The outer edge of the growth module comprises a plurality of afirst securing or fastening means or components, represented by 138a,b,c,d, extending therefrom that secure the growth module to thenutrient module at the lip, rim or edge 141 a comprising the proximalend 141 of the nutrient module. The outer edge of the growth module alsocomprises a plurality of a second means, generally represented by 139a,b,c,d, for securing or fastening the growth module to the nutrientmodule, such as snaps, clips or claimp, that are disposed between theprimary securing means. The combination of the first and second securingmeans forms a watertight seal between the modules.

With continued reference to FIGS. 2A-2B, FIG. 3 illustrates how thegrowth module 130 is fastened or secured to the nutrient module 140. Thegas port or vent 145 and its disposition in relation to thegas-permeable membrane 146 is depicted. One can see that upon fasteningthe modules together, the baffling system 136 and semi-permeabledialysis membrane 137 in the growth module are in fluid contact with thegas port 145 and gas-permeable membrane 146 in the nutrient module. Thefastening means 138 a,b,c,d comprise raised beveled edges 139 a,b,c,dwhich can slide or snap over the rim 141 a in the nutrient module along160 a,b to secure the growth module therein.

The following example(s) are given for the purpose of illustratingvarious embodiments of the invention and are not meant to limit thepresent invention in any fashion.

Example 1 Preparation of Alternating Ionic Magnetic Resonance CultureSystem Preparation of Cells for Culture/Medium

Under sterile conditions, a seeder culture is started with about 35 to50 ml of a human or mammalian cell suspension containing approximately1×10⁵−5×10⁶ cells/ml in a 50 ml cell culture flask.

The conditions of growing cells in a high density environment requiresthe use of a high quality media having a minimal concentration ofglucose at 4 g/l, and a sufficient buffer component such as NaHCO₃ asfound in standard media (3.7 g/l) which generally provides bufferingcapacity for a period of up to 2-10 weeks under standard cultureconditions. Because the culture system enables high-density growth, thebuffer is generally changed 1 to 2 times per week. The medium in thenutrient module should be replaced as soon as the color starts to changefrom salmon-pink to a yellowish-pink. Due to the high-density growthinside the growth module, the media will tend to maintain a yellowishcolor once a critical mass is achieved.

Serum concentration is often critical when eukaryotic cells are grown athigh density and should be minimally maintained at levels normally usedin stationary culture, approaching 0-80% depending on the cell type.When cells are cultured for the production of secretory molecules, e.g.antibodies, serum concentrations should be 5-30% inside the growthmodule. Serum concentrations in the nutrient module can often be reducedbut should be tested with each individual cell type. Because the use ofserum can create foaming problems in the culture system environment, anantifoam agent may be used. Cell types that can be grown without serumshould be adapted for such growth prior to growth in the bioreactor.

Adherent cells, such as CHO, HEK 293, BHK, will generally grow first insuspension and then as aggregates. Some adherent cell types will producesecreted products more effectively if grown in the presence of amicrocarrier type bead to minimize large aggregates of cells and tooptimize cell surface (secretory) area.

Preparation of the Culture System

The culture system is assembled under sterile conditions, preferably ina sterile hood, by attaching to or slipping a pre-sterilized disposablegrowth module made of polycarbonate inside a reusable nutrient moduleabout ten times larger than the growth module. The growth module issupplied as a pre-sterilized disposable unit pre-packaged in a sterileblister pack and is pre-fitted with a sterile cap. It is inserted intothe sterile nutrient module fitted with internal guides to hold thegrowth module whereby the growth module snaps tightly into place with aliquid tight seal in the specially designed opening. The nutrient modulehas a separate media opening for periodic exchange of media in thenutrient module which is vented during assembly to vent displaced air.The reusable/disposable nutrient module is comprised of polycarbonate,and sterilized by autoclaving to a maximum of 121° C. for 30 min insidean autoclave bag before assembly or may be gamma sterilized.

Filling the Culture System

The filling steps are done with all of the equipment and solutionsequilibrated at the culture temperature to minimize condensation and gasexpansion or contraction in the system after assembly. The seederculture/cell suspension is introduced into the 35 ml growth module witha syringe or pipette through a fill port in the growth module cap takingcare to allow for venting of displaced air. This growth module cap hastwo ports with snap caps comprising rubberized septum caps for syringeinoculations and air removal. One port has a Luer Lock adapter thatpermits easy filling by a syringe, while the other port is opened toallow air to escape during filling. The growth module is filledcompletely with the seeder culture and an appropriate media, removingall air from the growth module. The ports are sealed and last traces ofair are removed by insertion of an empty syringe with a needle into therubberized snap caps and withdrawing all air.

The nutrient module is filled to almost full capacity with about 450-500ml nutrient medium through the inlet port while removing air pressurethrough the gas-permeable silicone membrane in the nutrient module. Asmall air space is maintained to provide exchange of gases through thegas port. The inlet port is tightly sealed.

Applying the Electromagnetic Chamber

Once the modules are assembled, filled and sealed, the removableelectromagnetic chamber, fitted to the diameter of the nutrient moduleis slipped over the entire unit from the end distal FIG. 1C to the endwith the filling caps and ports. A flexible swivel cord adapter on thechamber enables the entire unit to rotate without interference from theelectrical cord that supplies the current to generate the pulsatingelectromagnetic field. The chamber imparts a time-varyingelectromagnetic force (square/delta wave, Fourier curve) to the culturesystem growth chamber and its contents.

Applying the Gravity Vector Randomization Device

The assembled culture system is placed on a gravity vector randomizationdevice inside an incubator chamber set at the temperature adapted forthe specific cell culture, which in this case is 35-37° C. and set torotate the human hybridoma cells at about 5 rpm. The culture system ismonitored for leaks and other problems and incubated while continuallyrandomizing the gravity vector until the first sample is taken.Different mammalian cell types require different randomization rates.For example, murine hybridoma cells are generally grown at 5 to 20 rpm,whereas human and transfected cells do well with slightly fasterrotation rates of about 10 to 100 rpm. These cell lines are not intendedto be limiting to the current invention as the current culture system isintended to be adaptable for the growth of any cell type or tissues thatcan be adapted to traditional cell culture methods.

Taking Samples and Harvesting

Samples are periodically taken from the culture system in order toassess the growth and development of the cultured material. Beforetaking samples, the culture system is taken from the incubator, removedfrom the continually randomized gravity vector device and theelectromagnetic chamber is removed. All steps are done quickly tominimize settling of the cells. The culture system is then wiped down tominimize contamination and transferred to a sterile hood. Inside thehood, built up pressure is released by slowly opening the media port inthe nutrient module. The growth module can then be sampled by openingthe fill port with the Luer lock adapter. The volume removed is replacedwith an equal volume of fresh media and the chamber is resealed,reassembled with the electromagnetic chamber and reset on thecontinually randomized gravity vector device in the incubator chamber.

Changing the Medium in the Nutrient Module

Replacing the spent medium with fresh medium should be done about 1-2times per week and requires dismantling of the culture system in thesame manner as if taking a sample, but the growth module is leftunopened. Instead, the nutrient module fill cap is removed and the usedmedium is emptied by carefully pouring out the contents in a sterilehood. About 350 to 400 ml of fresh media (37° C.) is poured into thenutrient module and reassembled as before. Care is taken to minimize anycontamination of the modules or media.

Cell Culture Density

Growth of high-density cells, such as hybridoma cells, requires moreoxygen, more nutrients and more frequent removal of waste products andis therefore better accommodated with a continual flow design nutrientmodule. For example, the oxygen requirement of hybridoma cells at about10⁷ cells/ml in a 35-50 ml growth module is about 1.75 mg/hr. Some celllines do not grow to high densities (less than 2×107 cells/ml) but maybe cultivated for a longer period of time in the culture system forproduction and harvest of secreted products with regular changes ofmedium or a continual flow nutrient module.

Production of Secreted Cell Products

Cell products such as monoclonal antibodies, cytokines, pro-inflammatorymolecules, biomolecular markers and all other soluble biochemicalproducts can be produced in a culture system once the cells have beencultured to a critical cell density which depends on the individualproperties of the cells cultured. Hybridoma cells typically producebetween 4×10⁷ and 7×10⁸ antibody molecules per cell in a 24-hour period.

Genomic Analysis of Tissue-Like Assemblies (TLAs)

RNA from tissue-like assemblies of cells grown in GTSF-2, RPMI 1640,Hams F10, MEM Alpha, L-15, Dulbecco's Modified Eagles Medium (DMEM),Hams F12, Earls MEM, DMEM/F12, or other media appropriate to the celltype with out without alternating ionic magnetic resonance was harvestedby removing it from the 3D device and placing in a 50 ml tube. Media wasremoved and the tissue-like assemblies were washed 3× with sterile PBS.After washing the tissue-like assemblies were frozen at −80 C. andstored for transfer to Asuragen Inc. Samples were sent to Asuragen fordigestion of the RNA and gene array chip analyses on Affymetrix U133 2.0plus human genome chips. Digital Chip data was sent to the laboratoryand processed by analyses in Genspring software.

3D TLA Growth Kinetics and Glucose Consumption

Metabolic parameters of tissue-like assemblies were measured every 24-48h over the course of the experiments to monitor a cellular developmentprofile and to monitor the metabolic status of the tissues. Glucoseconsumption was determined using the iStat clinical blood gas analyzerusing an EC8⁺ cartridge (Abbott Laboratories, Abbott Park, Ill.)according to the manufacturer's instructions (1).

Example 2 Gene Induction in HBTC Cells Grown in the Alternating IonicMagnetic Resonance Culture System HBTC TLA 3D Cell Culture

A culture of cells comprising fibroblasts, mesenchymal and secretorycells (HBTC) were cultured in the alternating ionic magnetic resonanceculture system. A mixture of human bronchi and tracheae primary cells(HBTC; fibroblasts and mesenchymal cells) were obtained from the lungmucosa of multiple tissue donors through Cambrex Biosciences(Walkersville, Md.) and were shown to be free of viral contamination bya survey of a panel of standard adventitious viruses (e.g. HIV,hepatitis, herpes) conducted by the supplier (Cambrex). The cells wereinitially grown as monolayers in human fibronectin coated flasks (BDBiosciences, San Jose, Calif.) and propagated in GTSF-2 mediasupplemented with 10% fetal bovine serum (FBS). GTSF-2 media, initiallydescribed in U.S. Pat. No. 5,846,807, is a tri-sugar-based growth mediumcontaining glucose, galactose and fructose. U.S. Pat. No. 5,846,807 isherein incorporated by reference in its entirety.

The monolayers were grown in a Form a humidified CO₂ incubator with 95%air and 5% CO₂ at constant atmosphere and at 37° C. The HBTC cells werepassaged using enzymatic dissociation with a solution of 0.1% trypsinand 0.1% EDTA for 15 minutes at 37° C. After incubation with theappropriate enzymes, the cells were transferred to 50 ml Corning conicalcentrifuge tubes and centrifuged at 800 g for 10 minutes. The pelletedcells were suspended in fresh GTSF-2 medium and diluted into T-75 flasksusing 30 ml of fresh growth medium.

The culture assembly was inoculated with HBTC (mesenchymal) cells andgrown for several weeks. HBTC cells were first removed from the T flasksby enzymatic digestion, washed once with calcium- and magnesium-freephosphate-buffered saline (CMF-PBS), and assayed for viability by trypanblue dye exclusion (Gibco). Cells were held on ice in fresh growthmedium prior to inoculation of the culture assembly. The primaryinoculum for the culture experiment included 2×10⁵ cells/ml HBTC cells,which were added to fresh GTSF-2 media in a 35-ml growth module with 5mg/ml of Cytodex-3 (Type I, collagen-coated cyclodextran) microcarriershaving a diameter of 120 mm (Pharmacia, Piscataway, N.J., USA). The 450ml nutrient module was filled with fresh GTSF-2 media, the cultureassembly was sealed as described above.

Briefly, the alternating ionic magnetic resonance is supplied to theculture unit by encompassing the culture assembly with the removable andadjustable alternating ionic magnetic resonance coil. At increasingfrequencies cultured cells and media are exposed to an alternating ionicmagnetic resonance signal at fundamental intervals of 10, 14, 15, 16 and32 Hz including the harmonic intervals of each of these extending to 256Hz and incorporating all harmonics of the aforementioned fundamentalfrequencies to infinity in the form of a square wave of 0.01-5000 mA.The alternating ionic magnetic resonance chamber providing theelectromagnetic protocol was placed around the culture device and aseries of stepped resonance pulses at approximately 500 msec intervalswas applied to the outside of the culture assembly. The culture assemblyand the unit was connected to a continuously randomized gravity deviceand grown in a Form a humidified CO₂ incubator with 94.5% air and 5.5%CO₂ providing constant atmosphere at 35.0° C. to mimic that of thenasopharyngeal epithelium. The HBTC cultures were allowed to grow for aminimum of 24 hours before the medium was changed. Thereafter, freshmedium was replenished by replacing 65-100% of the spent medium withinthe nutrient module once every 96-168 hour period.

At this point, the media for the culture experiments comprised GTSF-2supplemented with 10% fetal bovine serum. As the cells proliferated,metabolic requirements increased, and the fresh medium was routinelysupplemented with an additional 100 mg/dl of glucose.

The culture was sampled periodically over the course of the experiment,generally at 24-48 hour time points, in order to establish a cellulardevelopment profile. The parameters of glucose utilization (FIG. 5A) andpH (FIG. 5B) were surveyed via iStat™ clinical blood gas analyzer todetermine the relative progress and health of the cultures and the rateof cellular growth and viability.

The cells were monitored as shown in FIGS. 6A to 6D. FIG. 6A showsphotos of the HBTC cells grown in T-flasks (passaged as necessary tomaintain growth over a 20-day period) that have been infused with thecalcium binding fluorescent dye, Fura-2AM. The cells on the left wereexposed to multi-variant electromagnetic frequency field for theentirety of the 20-day growth period. Cells in the right panel were notexposed to an electromagnetic field. Exposure to alternating ionicmagnetic resonance altered the cellular distribution of calcium ions.FIG. 6B shows HBTC cells grown on cultisphere or Cytodex-3 microcarriersin the alternating ionic magnetic resonance bioreactor for 21 days. Theupper left panel shows cells exposed to alternating ionic magneticresonance for the entire growth period; the cells on the right were notexposed to an electromagnetic field. Tissue-like assemblies of cells andmicrocarrier beads (tissue-like assemblies) were treated with Fura-2AM.Although the microcarriers shown retain background levels offluorescence as evidenced by the photo in the bottom control panelshowing the microcarrier alone, the cells exposed to alternating ionicmagnetic resonance emit a significant signal above background levels.Arrows indicate clusters of cells attached to, but not atop amicrocarrier bead. FIG. 6C is similar to cells shown in FIG. 6A, withHBTC cells grown with (left) or without (right) alternating ionicmagnetic resonance, but infused with the potassium binding fluorescentdye, PBFI-2 AM. FIG. 6D illustrates potassium ion staining of cellsgrown in the presence of microcarrier beads either with (top) or without(middle) alternating ionic magnetic resonance compared to a microcarrieralone control treated with PBFI-AM (bottom).

Gene Induction in the Alternating Ionic Magnetic Resonance-Grown HBTCTLAs

HBTC cells grown in the alternating ionic magnetic resonance culturesystem demonstrate up-regulation of genes within specific gene families,including levels of expression for various transport and regenerativegenes. Table 1 lists genes up-regulated in an alternating ionic magneticresonance field and provides the fold increase relative to level of geneexpression in cells grown without a magnetic resonance field.

TABLE 1 Alternating Ionic Magnetic Resonance Initiated GeneUp-Regulation GENE FAMILY FOLD GENE SYMBOL GENE NAME INCREASE TRANSPORTFAMILY GENES (1) Calcium Ion Transport KCNMB1 Potassium largeconductance +62 Calcium-activated channel, subfamily M, beta member 1CABP1 calcium binding protein 1 +52 (calbrain) CACNG1 calcium channel,voltage- +46 dependent, gamma subunit 1 CABP2 calcium binding protein 2+36 SLC24A3 solute carrier family 24 +12 (sodium/potassium/calciumexchanger), member 3 CACNA1C calcium channel, voltage- +3.6 dependent, Ltype, alpha 1C subunit TACSTD tumor-associated calcium signal +2.8transducer 2 CACNB1 calcium channel, voltage- +7.3 dependent, beta 1subunit CALB3 calbindin 3, vitamin D-dependent +4.2 calcium bindingprotein KCNMA1 potassium large conductance +3.0 calcium-activatedchannel, subfamily M, alpha member 1 CACL2 chloride channel, calcium+2.5 activated, family member 2 CAMK2A calcium/calmodulin-dependent +2.5protein kinase (CaM kinase) II alpha S100A5 S100 calcium binding proteinA5 +2.3 (2) Potassium IonTransport KCNMB1 Potassium large conductance+61 Calcium-activated channel, subfamily M, beta member 1 KCND3potassium voltage-gated +21 channel, ShaI-related subfamily, member 3SLC24A3 solute carrier family 24 +8.6 (sodium/potassium/calciumexchanger), member 3 KCNK15 potassium channel, subfamily K, +3.7 member15 KCNK3 potassium channel, subfamily K, +2.1 member 3 KCNJ12 potassiuminwardly-rectifying +7.6 channel, subfamily J, member 12 KCNQ1 potassiumvoltage-gated +3.3 channel, KQT-like subfamily, member 1 KCNAB1potassium voltage-gated +3.1 channel, shaker-related subfamily, betamember 1 KCND3 potassium voltage-gated +3.0 channel, ShaI-relatedsubfamily, member 3 KCND2 potassium voltage-gated +2.8 channel,ShaI-related subfamily, member 2 SLC12A5 solute carrier family 12, +2.7potassium-chloride transporter member 5 KCNJ5 potassiuminwardly-rectifying +2.5 channel, subfamily J, member 5 SLC24A1 solutecarrier family 24 +2.2 (sodium/potassium/calcium exchanger, member 1KCNE1L potassium voltage-gated +2.2 channel, Isk-related family, member1-like KCNK7 potassium channel, subfamily K, +2.0 member 7 KCNK4potassium channel, subfamily K, +1.9 member 4 (3) ATPase TransportATP6V0A4 ATPase, H+ transporting, +32 lysosomal V0 subunit a isoform 4ATP2A3 ATPase, Ca++ transporting, +14 ubiquitous ATP1A2 ATPase, Na+/K+transporting, +6.5 alpha 2 (+) polypeptide SERCA3 ATPase, Ca++transporting, +6 ubiquitous ATP2A1 ATPase, Ca++ transporting, +13cardiac muscle, fast twitch 1) ATP6V0A2 ATPase, H+ transporting, +7.7lysosomal V0 subunit a isoform 2 ATPase mRNA sequence +7.2 ATP11AATPase, Class VI, type 11A +5.6 ATP-binding Cassette, sub-family A(ABC1), +4.7 member 6 TAP2 transporter 2, ATP-binding +4.5 cassette,sub-family B (MDR/TAP) ABCC9 ATP-binding cassette, sub-family +2.7 C(CFTR/MRP), member 9 ATP4B ATPase, H+/K+ exchanging, beta +2.5polypeptide ATP8A1 ATPase, aminophospholipid +2.5 transporter (APLT),Class I, type 8A, member 1 ABCA8 ATP-binding cassette, sub-family +2.4 A(ABC1), member 8 ATP6V1B1 ATPase, H+ transporting, +2.4 lysosomal 56/58kDa, V1 subunit B, isoform 1 ATP5G2 ATP , H+ transporting, mitochondrial+2.0 synthase F0 complex, subunit c (subunit 9), isoform 2 ATP1B4ATPase, (Na+)/K+ transporting, +1.9 beta 4 polypeptide REGENERATIONGENES (1) WNTs Family WNT2 wingless-type MMTV integration +21038 sitefamily member 2 WNT16 wingless-type MMTV integration +180.8 site familymember 16 WNT3 inducible signaling pathway +45.8 protein 3 WNT4 +78.8WNT8B +3.3 WNT1 +1.7 (2) Bone Morphogenetic Protein Family (BMP2) BoneMorphogenetic Protein 2 +166.6 (BMP5) Bone Morphogenetic Protein 5 +88.0BMP-2 inducible BMP inducible kinase +5.5 kinase BMPY Bone MorphogeneticProtein Y +2.4 (BMP6) Bone Morphogenetic Protein 6 +1.6 (3) CateninFamily Catenin cadherin-associated protein, +38.3 delta 2 (neuralplakophilin-related arm-repeat protein) Catenin +2.1 (4) Forkhead BoxFamily FOXI1 Forkhead Box I1 +60.4 FOXA2 Forkhead Box A2 +9.0 FOXD1Forkhead Box D1 +3.4 FOXA1 Forkhead Box A1 +2.0 FOXM1 Forkhead Box M1+1.6 (5) SOX (SRY (Sex Determining Region Y)-BOX2) Family SOX2 +2.2 SOX3+1.9 SOX29 +1.8 SOX17 +1.7 (6) Transforming Growth Factor (TGF) FamilyTGFBR2 transforming growth factor, beta +1.9 receptor II TGFAtransforming growth factor, alpha +1.9 TGIF2 TGFB-induced factor 2 (TALE+1.7 family homeobox) (7) Parathyroid Hormone (PTH) Family PTHparathyroid hormone +180 PTHLH parathyroid hormone-like +1.8 hormonePTHR2 parathyroid hormone receptor 2 +1.6

Example 3 Gene Induction in NHNP and HBE Cells Grown in the AlternatingIonic Magnetic Resonance Culture System NHNP TLA 3D cell culture

NHNP cells were obtained from Lonza (Walkersville, Md., USA) andpropagated in GTSF-2, a unique media containing glucose, galactose andfructose supplemented with 10% fetal bovine serum (FBS), at 37° C. undera 5% CO₂ atmosphere (2-4). NHNP cells were initially grown as monolayersin human fibronectin-coated flasks (BD Biosciences, San Jose, Calif.)and pooled from at least five donors, as described previously (5). NHNPcell cultures were expanded, tested for viral contaminants aspre-certified by the manufacturer's production criteria (Lonza), andcryopreserved in liquid nitrogen. Three-dimensional (3D) NHNP TLAs weregenerated by seeding 3×10⁵ NHNP cells/ml onto 3 mg/ml Cultispher beads(Sigma-Aldrich, St. Louis, Mo.) into a 55 ml rotating wall vesselbioreactor (RWV; Synthecon, Houston, Tex.) or into the culture unit ofthe alternating ionic magnetic resonance culture apparatus and grown at37° C. under a 5% CO₂. Cells were allowed to attach to the beads for 48h in the bioreactor before re-feeding with GTSF-2 containing 10% FBS. Tomaintain the TLA cultures within normal human physiological bloodchemistry parameters (pH 7.2 and a glucose concentration of 80-120mg/dL), 20-90% of the media was replaced as required with fresh GTSF2media every 48 h, facilitating efficient tissue-like assembly tissuegrowth and maturation prior to VZV infection. All metabolicdeterminations were made using an iStat hand held blood gas analyzer(Abbott Laboratories, Abbott Park, Ill.). Flow cytometry analysisconfirmed that after 180 days in culture, NHNP TLAs expressed neuronalprogenitor markers CXCR4, CD133, CD105-Endoglin, CD 90-Thy-1 andCD49f-α6 Integrin at levels comparable to a parental NHNP (2D) cellpopulation.

HBE TLA 3D Cell Culture

Mesenchymal cells (HBTC) from human bronchi and tracheae were obtainedfrom three donors through Cambrex Biosciences (Walkersville, Md.).LLC-MK2 and BEAS-2B epithelial cells (6) were obtained from ATCC(Manassas, Va.). BEAS-2B cells were used instead of primary cells toprovide consistency from batch to batch. BEAS-2B and HBTC cells weremaintained in GTSF-2 medium with 7% fetal bovine serum (7) in humanfibronectin coated flasks (BD Biosciences, San Jose, Calif.). Vero,HEp-2, and LLC-MK2 cells were grown at 37° C. in Eagle's modifiedminimum essential medium supplemented with 2 mM non-essential aminoacids, 100 units penicillin, 100 μg/ml streptomycin, 0.25 μg/mlamphotericin B, 10% fetal bovine serum, 2 mM L-glutamine, and 25 mMHEPES buffer (Gibco-BRL, Gaithersburg, Md.).

To construct 3D HBE tissue-like assembly cultures, HBTC cells from amonolayer culture were seeded at 2×105 cells/mL into a 55-mL rotatingwall vessel (RWV) (Synthecon, Houston, Tex.) or into the culture unit ofthe alternating ionic magnetic resonance culture apparatus with 4-5mg/mL of Cytodex-3 microcarriers, type I collagen-coated cyclodextranmicrocarriers (Pharmacia, Piscataway, N.J.) at 35° C. Cultures wereallowed to grow for a minimum of 48 hours before the medium was changed.BEAS-2B cells were seeded at 2×10⁵ cells/mL 4 to 6 days afterHBTCCytodex 3 microcarrier aggregates were formed. Thereafter,approximately 65% of the media was replaced every 20 to 24 hours. Asmetabolic requirements increased, the glucose concentration in GTSF-2medium was increased to 200 mg/dL. Tissue-like assembly cultures weregrown in RWV to 1 to 2 mm in diameter using the rotary cell culturesystem (Synthecon, Houston, Tex.) or into the culture unit of thealternating ionic magnetic resonance culture apparatus at 35° C. withappropriate rotation rate for aggregate suspension. Cell numbers weredetermined after treating the tissue-like assemblies with 2000 U/mL typeI collagenase (Invitrogen, Carlsbad, Calif.) at 37° C. for 10 minutes.Expression levels of epithelial markers in TLAs are very similar to thelevels in normal human lung than in 2D BEAS-2B and HBTC cells.

Alternating Ionic Magnetic Resonance-Exposed HBE and NHNP TLAs

A set of human bronchial epithelial (HBE) tissue-like assembly samplesand a set of normal human neural progenitor (NHNP) tissue-like assemblysamples, each set contained in at least 3 rotating wall vessels (RWVs)were exposed to an alternating ionic magnetic resonance stimulationfield of predetermined profile. The profile substantially comprises abiphasic, square wave with a frequency of about ˜10 Hz, a wavelength ofabout 500 ms, a rising slew rate between about 0.1 T/s (1.0 kG/s) toabout 0.50 T/s (5.0 kG/s), a falling slew rate between about 0.50 T/s(5.0 kG/s) and about 2.0 T/s (20.0 kG/s), a dwell time of about 10%after each burst, a duty cycle of about 80% on and about 20% off, and aresultant B-Field magnitude of about 100 μT (1.0 G). The experiment wasconducted at ˜10 Hz. For reference purposes, the frequency of Earth'sgeomagnetic field is 7.83 Hz, thus the experiment satisfies the criteriaof being appreciably different from the background magnetic field.Exposure was continuous for the duration of a period of about 15-90 daysor about 360-2160 hours. A gene fold change analysis, as describe inExample 1, was conducted for the HBE and NHNP TLA samples exposed to thealternating ionic magnetic resonance stimulation field.

Gene Induction in Alternating Ionic Magnetic Resonance-Grown HBE andNHNP TLAs

Although the HBE and NHNP tissue-like assemblies responded differentlywhen exposed to an alternating ionic magnetic resonance field undersubstantially identical conditions, unexpectedly, in both setsvirally-associated and viral oncogenes were activated and theirexpression levels were upregulated. Most of the differentially regulatedgenes are related to the ability of viruses to be absorbed or introducedinto the human cell to enable replication and proliferation. Table 2lists the up-regulated viral genes initiated by alternating ionicmagnetic resonance.

TABLE 2 Alternating Ionic Magnetic Resonance Initiated Viral GeneUp-Regulation GENE SYMBOL GENE NAME FOLD INCREASE HBE TLA GENES RAB6ARAB6A virus associated 2.7991202 RAB5B RAB5B virus associated 1.5721819IVNS1ABP influenza virus NS1A binding protein 1.5685624 JUN jun oncogene1.6782385 JUNB jun B proto-oncogene 1.5822183 HTATSF1 HIV-1 Tat specificfactor 1 1.8373269 FOSB FBJ murine osteosarcoma viral oncogene homolog B1.5298892 JUND jun D proto-oncogene 3.2985632 CXADR coxsackie virus andadenovirus receptor 1.8511372 EVI2A ecotropic viral integration site 2A1.922923 EBI2 Epstein-Barr virus induced gene 2 (lymphocyte- 1.7887306specific G protein-coupled receptor) IVNS1ABP influenza virus NS1Abinding protein 1.7672802 AKT1 v-akt murine thymoma viral oncogenehomolog 1 1.5862192 FOS v-fos FBJ murine osteosarcoma viral oncogene4.55682 homolog UBE3A ubiquitin protein ligase E3A (human papillomavirus 1.5529021 E6-associated protein EVI2B ecotropic viral integrationsite 2B 1.566628 RRAS related RAS viral (r-ras) oncogene homolog1.6267682 HRB HIV-1 Rev binding protein 1.6248319 JUND jun Dproto-oncogene 2.2347317 FYN FYN oncogene related to SRC 1.8570358 ITPR1inositol 1 1.5927685 RAB23 RAB23 1.7071296 AKT3 v-akt murine thymomaviral oncogene homolog 3 1.8035016 (protein kinase B BIRC6 baculoviralIAP repeat-containing 6 (apollon) 1.5996437 THRB thyroid hormonereceptor 1.7461618 TPR translocated promoter region (to activated MET1.7921942 oncogene) NHNP TLA GENES ETS2 v-ets erythroblastosis virus E26oncogene homolog 1.5718486 2 (avian) JUNB jun B proto-oncogene 2.720869EGFR epidermal growth factor receptor (erythroblastic 2.6352847 leukemiaviral (v-erb-b) oncogene homolog BIRC5 baculoviral IAP repeat-containing5 (survivin) 1.6022671 FOSB FBJ murine osteosarcoma viral oncogenehomolog B 2.0152645 PVRL2 poliovirus receptor-related 2 (herpesvirusentry 1.6020694 mediator B) SKIV2L superkiller viralicidic activity2-like (S. cerevisiae) 1.520639 FOS v-fos FBJ murine osteosarcoma viraloncogene 5.8558683 homolog TNFRSF14 tumor necrosis factor receptorsuperfamily 1.8052368 member 14 (herpesvirus entry mediator) PVRpoliovirus receptor 2.9330401 THRA thyroid hormone receptor (Alpha(erythroblastic 1.5989169 leukemia viral (v-erb-a) oncogene homologMOV10 Mov10 Maloney Leukemia Virus 10 1.8294989 PVRL2 poliovirusreceptor-related 2 (herpesvirus entry 1.5439448 mediator B) ISY1 ///ISY1 splicing factor homolog (S. cerevisiae) /// 1.802375 RAB43 RAB43MRVI1 murine retrovirus integration site 1 homolog 2.84193 MAFK v-mafmusculoaponeurotic fibrosarcoma oncogene 1.6252148 homolog K (avian)RAB4B RAS oncogene family 1.5139388 MRVI1 murine retrovirus integrationsite 1 homolog 2.0572836 RAB7B RAS oncogene family 1.5806108 LOC401233similar to HIV TAT specific factor 1; cofactor 2.2209923 required forTat activation of HIV-1 transcription

Example 4 3-Dimensional TLA Models of Viral Infection v63G/70R Infectionof NHNP TLAs

Varicella zoster virus (VZV) was propagated in human melanoma cells(MeWo, American Type Culture Collection, ATCC, Manassas, Va.) inDulbecco's minimal essential medium supplemented with 10% fetal bovineserum, 100 U/ml penicillin and 0.1 mg/ml streptomycin (Sigma-Aldrich,St. Louis, Mo.) at 37° C. under a 5% CO₂ (8). Wild-type and recombinantviruses were passaged on MeWo cells by co-cultivation of infected withuninfected cells at a ratio of 1/5 (9). MeWo cells for the infection ofNHNP cultures were adapted to GTSF2 medium over two passages prior toharvest of VZV.

Cell free VZV was used for the NHNP TLA infections to avoid transfer ofany infected MeWo cells to the TLA culture (10). Briefly, infected cellswere harvested at 96 h post-infection (p.i.) and resuspended inreticulocyte standard buffer (10 mM NaCl, 1.5 mM MgCl₂, 10 mM Tris-HCl,pH 7.4). Cells were disrupted by Dounce (type A) homogenization(Cole-Parmer, Vernon Hills, Ill.) and clarified by centrifugation at900×g for 15 min.

NHNP tissue-like assemblies were infected in the RWV with cell-free VZVat a multiplicity of infection (MOI) of 0.1 by absorption at room tempfor 30 min in 20 ml GTSF-2. Then, the RWVs were filled with freshGTSF-2/10% FBS and transferred to a humidified incubator with under 5%CO₂ atmosphere at 37° C. Every 24 h p.i., 55-65% of the culture mediawas replaced with fresh GTSF-2 containing 10% FBS. Samples werecollected approximately every other day for ˜70 days to determine viralgenome copies utilizing a dually tagged v63G/70R recombinant.

Dually tagged v63G/70R was able to efficiently infect NHNP tissue-likeassemblies, as evidenced by an approximate 50-fold increase in VZVgenome copies from 0 to 18 days post-infection (dpi). After 18 dpi, VZVgenome copy numbers remained constant, indicating that the virus hadestablished equilibrium between de novo virus DNA replication anddegradation. Glucose utilization was used to monitor the metabolicactivity of infected and uninfected 3D NHNP tissue-like assemblies andMeWo cultures. Each culture was initially maintained for 39 days toestablish a baseline glucose consumption rate before and after infectionwith v63G/70R. Upon infection, glucose utilization rapidly declined inMeWo cells, as cell death, likely as a consequence of lytic VZVreplication, was evident microscopically (data not shown). In contrast,glucose uptake in NHNP tissue-like assemblies was not altered as aresponse to VZV infection, suggesting that limited, if any, lytic VZVinfection occurred. Confocal analysis of v63G infected NHNP tissue-likeassemblies at 27 dpi revealed that the progenitor neuronal marker Nestinand the mature neuronal marker β Tubulin-III colocalized with GFP,indicating that VZV preferentially established a persistent infection inthese cells.

GFP/RFP ratios in v63G/70R infected NHNP tissue-like assemblies remainedunaltered for at least 69 days in culture, suggesting that the VZVgenome is stably maintained in NHNP tissue-like assemblies. In addition,confocal microscopy confirmed that both ORF63-eGFP and ORF70-mRFP areexpressed in NHNP tissue-like assembly cultures infected with v63G/70R.A stable viral genome is preserved for an extended period in NHNPtissue-like assemblies.

Paramyxovirus Infection of HBE TLAs

Tissue-like assemblies were inoculated at a MOI of 0.1 with both wtPIV3and attenuated PIV3 viruses to achieve an effective MOI of 1 for thecells on the outer surface. After virus absorption at room temperaturefor 1 hour, HBE TLA cultures were washed three times with DPBS(Invitrogen, Carlsbad, Calif.) and fed with media. All air bubbles wereremoved from the RWV before rotation to eliminate shearing of the cells(7). Approximately 65% of the culture media was replaced every 48 hours.For virus titration, samples were collected on days 0, 2, 4, 6, 8, and10. In HBE tissue-like assemblies, replication of wtPIV3 approached 7.5log 10 pfu/mL by day 6 pi, while the attenuated virus replicatedmaximally to 5.5 log 10 pfu/ml on day six. Reduced replication inattenuated viruses may be due to a slower progression from layer tolayer in the 3D HBE tissue-like assemblies. Inceased secretion of atleast 2-fold of cytokines, chemokines and colony stimulating factors wasfound for interleukin-1, -4, -6, and -8, for RANTES, MIP-1a, MIP-1, andG-CSF.

THE FOLLOWING REFERENCES ARE CITED HEREIN

-   1. Vertrees et al. Cancer Biol Ther 8:356-365 (2009).-   2. Lelkes et al. In Vitro Cell Dev Biol Anim 33:344-351 (1997).-   3. Goodwin, T. J. U.S. Pat. No. 5,846,807 (1998).-   4. Goodwin, T. J. U.S. Pat. No. 5,858,783 (1997).-   5. Goodwin, T. J. Physiological and Molecular Genetic Effects of    Time-Varying Electromagnetic Fields on Human Neuronal Cells. NASA    Tech Paper, TP-2003-212054 (2003).-   6. Ke et al. Differentiation 38:60-66 (1988).-   7. Goodwin et al. Proc Soc Exp Biol Med 202:181-192 (1993).-   8. Grose et al. J Gen Virol 43:15-27 (1979).-   9. Cohrs et al. J Virol 76:7228-7238 (2002).-   10. Grose et al. Infect Immun 19:199-203 (1978).

While the present invention is described with reference to one or moreparticular embodiments, those skilled in the art will recognize thatmany changes may be made thereto without departing from the spirit andscope of the present invention. Each of these embodiments and obviousvariations thereof is contemplated as falling within the spirit andscope of the claimed invention set forth in the following claims.

What is claimed is:
 1. A method for culturing cells, comprising thesteps of: introducing cells into a culture unit having a growth moduleand a nutrient module; randomizing continually the gravity vector of thegrowth module; and applying a pulsating alternating ionic magneticresonance field to the growth module during culturing of the cells. 2.The method of claim 1, wherein the introducing step comprises: placingthe cells into the growth module; filling the growth module with growthmedia; sealing the growth module to remove observable gases; attachingthe growth module to the nutrient module; and adding fresh growth mediumto the nutrient module.
 3. The method of claim 1, wherein therandomizing step comprises: positioning the nutrient module comprisingthe culture unit into a randomizing adapter; and randomizing the gravityvector via a randomizing mechanism comprising the randomizing adapter.4. The method of claim 1, wherein the applying step comprises:positioning the growth module comprising the culture unit into anelectromagnetic chamber; and generating the pulsating alternating ionicmagnetic resonance field in the electromagnetic chamber.
 5. The methodof claim 1, further comprising the step of: modulating the alternatingionic magnetic resonance field to produce overlapping or fluctuatingalternating ionic magnetic resonance frequencies at one or more modalintervals spanning about 6.5 Hz and ranging from about 7.8 Hz to about59.9 Hz.
 6. The culture system of claim 5, wherein the overlapping orfluctuating alternating ionic magnetic resonance frequencies producedare about 10, 14, 15, 16, or 32 Hz.
 7. The method of claim 1, whereinthe nutrient module comprises: an open-ended body having a proximal endwith a diameter of a length to receive the growth module therein; afirst gas-permeable membrane with a gas port disposed thereon comprisingthe proximal end; and a distal end comprising a first sealable opening.8. The method of claim 7, wherein the growth module comprises: a bodyhaving a proximal end comprising a second gas-permeable membrane with aplurality of inlet/outlet ports disposed thereon; and a distal endcomprising a baffling system and a semi-permeable membrane in fluidcontact with the first gas-permeable membrane.
 9. The method of claim 1,wherein the growth module and the nutrient module are disposable. 10.The method of claim 1, wherein the alternating ionic magnetic resonancefield produces one or more of a modification, growth, differentiation,dedifferentiation, altered gene expression, altered transcription eventsor cellular regeneration via controlling the ionic transport ortranscription mechanism in the cells.
 11. The method of claim 1, whereinthe alternating ionic magnetic resonance field changes the mammaliancellular ionic transport thereby enabling infection and an expression ofmammalian viral epitopes.
 12. The method of claim 1, wherein the cellscomprise a tissue, an organoid body, a virally-infected cell, or abacterially-infected cell.
 13. A culture method for growing mammaliancells or tissues, comprising the steps of: introducing mammalian cellsor tissues into an alternating ionic magnetic resonance cultureapparatus comprising: a nutrient module, having a proximal end and asealable distal end, that contains a nutrient media; a growth module,having a proximal end and a distal end, that contains the cells ortissue and that is filled with nutrient media and sealed to removeobservable gases, said distal end of the growth module fluidly sealed tothe proximal end of the nutrient module; a randomizing adapterelectrically connected to a randomizing mechanism and containing thenutrient module therein; and an electromagnetic chamber comprising aconductive wire to produce a pulsating alternating ionic magneticresonance field; randomizing, continually, the gravity vector of thegrowth module with the randomzing adapter; and generating the pulsatingalternating ionic magnetic resonance field around the growth moduleduring culturing.
 14. The method of claim 13, further comprising thestep of: modulating the alternating ionic magnetic resonance field toproduce overlapping or fluctuating alternating ionic magnetic resonancefrequencies at one or more modal intervals spanning about 6.5 Hz andranging from about 7.8 Hz to about 59.9 Hz.
 15. The method of claim 14,wherein the overlapping or fluctuating alternating ionic magneticresonance frequencies produced are about 10, 14, 15, 16, or 32 Hz. 16.The method of claim 13, wherein the nutrient module comprises: a firstgas-permeable membrane with a gas port disposed thereon comprising theproximal end.
 17. The method of claim 16, wherein the growth modulecomprises: a second gas-permeable membrane with a plurality ofinlet/outlet ports disposed thereon comprising the proximal end; and abaffling system and a semi-permeable membrane comprising the distal endin fluid contact with the first gas-permeable membrane.
 18. The methodof claim 13, wherein the growth module and the nutrient module aredisposable.
 19. The method of claim 13, wherein materials comprising thealternating ionic magnetic resonance culture apparatus are sterilizable.20. The method of claim 13, wherein culturing in the alternating ionicmagnetic resonance field produces one or more of a modification, growth,differentiation, dedifferentiation, altered gene expression, alteredtranscription events or cellular regeneration via controlling the ionictransport or transcription mechanism in the cells or tissues.
 21. Themethod of claim 13, wherein culturing in the alternating ionic magneticresonance field produces a change in the mammalian cellular ionictransport thereby enabling infection and an expression of mammalianviral epitopes.
 22. The method of claim 13, wherein the mammalian cellsor tissues are virally or bacterially infected.
 23. A model comprising atissue-like assembly of the cultured mammalian cells or tissue producedby the method of claim 13.